Multi-Center Trial of a Standardized Battery of Tests of Mouse Behavior A project funded by the NIH Office of Behavioral and Social Science Research via NIAAA and NIDA. Investigators: John Crabbe, Doug Wahlsten, Bruce Dudek Web site:

Staff

Goals of the Study Evaluate mouse phenotypes at three sites using simultaneously performed identical procedures. Establish the feasibility of implementing precise test protocols in multiple sites. We sought to maximize standardization of protocols across site, often to a level of minor nuance, and at times perhaps not the “best” methodology possible for a task. Examine stability of strain differences across three laboratories, shipping status, and gender. Evaluate a broad-based behavioral test battery. Generate a roadmap for future studies designed to focus on narrower domains of phenotypes.

GROUPING FACTORS Genotype (Stock) A/J C57BL/6J BALB/cByJ DBA/2J 129/SvEvTac 5HT1B++ 5HT1B-- B6D2F2 Site Albany Edmonton Portland Sex Female Male Shipping Status Bred In House Shipped

Thus the design is an 8 x 3 x 2 x 2 factorial. 96 cells. Targeted n=4/cell (384 planned), based on power analysis. 379 tested, 378 completely. No empty cells Design Specifications

Replications Testing protocol required replications. Two replications yielded n=64 per site per replication. This produced a target of n=2 per cell across all cells in each replication. One site ran a third replication to fill some cells (n=6).

Chronology Breeding stock received 12/2/97 Matings set 1/13/98 First litters born ~ 2/1/98 Shipped mice for testing received 3/18/98 to 4/2/98 First replication begun 4/20/98 Second replication begun 4/27/98 Third replication begun 5/4/98 (Edmonton Only) Age at testing initiation: ~ days

Features of Husbandry Equated Across Labs Four time frames may differ in the requirements to some extent. 1. After arrival from supplier but before mating: housed with same sex 2. Mated pairs before and after delivery of litter 3. Weaned offspring prior to the start of behavioral testing 4. During Behavioral Testing After arrival of Breeding Stock FoodPurina 5001 (4.5% fat) BeddingBed-O-Cob 1/4" Nesting materialNone Cage topStainless steel bars (passive filter top in Portland) Animal markingnil Animal weighingnil Mice/cage4 or 5 maximum Lights on/off0600/1800 Cage changingweekly

Features of Husbandry Equated Across Labs, cont’d Mated pairs (items the same as after arrival are not repeated) FoodPurina 5020 (9% fat) Nesting material1 Nestlet Markingnone Weighingat mating Mice/cage1 female, 1 male Male present with litter?Yes; postpartum pregnancy allowed Inspection of new litterCount live and dead pups; remove dead, put live in nest Culling of newbornsRemove only the obvious runts for ethanasia Cage changingWeekly Cage changing with litterDo not disturb cage until litter is 4 days old or younger; then clean cage with fresh nestlet

Features of Husbandry Equated Across Labs, cont’d Weaned offspring Age at weaningAim for 21 days; may be ± 1 day on weekend Nesting materialNone FoodPurina 5001 Mice/cageSame sex, 4 or 5 maximum Markingnil Body WeightRecord at weaning to closest.5g During behavioral testing Mice/cage2 (housed 2 weeks prior to testing; random culling) Nesting materialNone MarkingSharpie pen on tail Weighingprior to day 5 activity test HandlingHand with surgical glove Cage changingAfter Day 5 testing

Targeted Phenotypic Domains Reproductive performance Basic growth characteristics Behavioral test battery Basic neuroanatomical characteristics

Variables Matched Across Site Chronology for breeding stock receipt, matings, receipt of shipped mice, and test battery beginning dates. Food, bedding, light cycle. Details of Behavioral Test Battery Protocols! Apparatus.

Behavioral and Other Phenotypes Locomotor Activity. 15 min test in Accuscan Digiscan system. Elevated Plus maze. 5 min test. Rota-rod coordination test. Water Maze escape task. Cocaine-stimulated locomotor activity. Alcohol preference. Neuroanatomical indices.

Test Battery Sequence Day 1: Baseline Locomotor Activity in Accuscan Digiscan automated activity monitors. (15 min test) Day 2: Elevated plus maze test. (5 min test) Day 3: Rota-rod test. (10 trials) Day 4: Water Escape test. (8 trials) Day 5: Cocaine-treatment activity test. (15 min test; 20 mg/kg, ip) Day 6-7: Null Day 8: Begin alcohol drinking test. (6 days)

Protocol Implementation Morning and afternoon sets of n=32 mice (16 cages) were run to complete the n=64/replication. Semi-blind testing was employed by using cage numbers rather than strain identity as identifiers (caveat regarding coat color). Testing order (which mouse genotype,sex, etc) was based on a constrained randomized sequence (although cage-pair mates were always tested in adjacent positions). This sequence varied across site. Same daily test routine for each squad of 2 or 4 mice was used on the first 5 tests. Documents describing protocols are available on the web site.

Current Status of Data Analysis Database for combined sites constructed, and web site created for data sharing. Database includes data from baseline activity test, rota- rod test, water escape test, cocaine activity test, elevated plus maze test, alcohol preference, and adult body weights. Statistical analyses of all tasks has been done and figures are available on the web site. An initial report of findings is published: Crabbe, Wahlsten and Dudek (1999) Science, 284:xx-xx.

General Conclusions From Present State of Data Analysis For the baseline activity, relatively good stability of the genetic differences is seen across site with virtually no meaningful influence of shipping status. Cocaine stimulated activity does vary considerably across site, but in an interactive manner with genotype. Strong patterns of genetic influence in the water escape task are only partly consistent across site and shipping status. Strain differences in alcohol consumption are relatively stable across site although a main effect of site is present. Elevated Plus Maze indices show considerably less cross site stability. The cross-site stability of strain differences is thus likely to be highly task-dependent.

Highlights of Lessons from the Multi-Center Standardization Trial 1. High quality data can be obtained simultaneously in several labs. 2. Logistics are much more challenging than for the typical study done in one lab. 3. Principal glitches can be avoided in future studies. 4. Anomalies or accidents afflicting one lab do not undermine the entire study. 5. a. A large advantage is conferred by the use of automated data collection. b. Testing $ 2 mice at one time is very difficult without automated apparatus. c. Video tape recording for later scoring from tapes is a markedly inferior method.

Highlights of Lessons from the Multi-Center Standardization Trial (cont’d) 6. We need to know more about the reliability of each test in order to plan future studies. 7. We need to standardize the stimulus surroundings for each test, not just the apparatus. 8. We should decide in advance what statistical analysis program will be used. 9. In the future, we should strive for optimal rather than convenience solutions.

Arrangement of apparatus in one lab